Use of TNF-alpha Inhibitors for Treating a Nerve Disorder Mediated by Nucleus Pulposus

ABSTRACT

The present invention relates to a method and a pharmaceutical composition for treatment of nerve disorders comprising administration of a therapeutically effective dosage of at least two substances selected from the group consisting of TNF inhibitors, IL-1 inhibitors, IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors, and IFN-gamma inhibitors. Preferably, at least one of the substances is a TNF inhibitor.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.11/521,093, filed Sep. 14, 2006, which is a continuation-in-part of U.S.patent application Ser. No. 10/225,237, filed on Aug. 22, 2002, now U.S.Pat. No. 7,115,557, which is a continuation in part of Ser. No.09/826,893 filed Apr. 6, 2001, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 09/743,852filed Jan. 17, 2001, now U.S. Pat. 6,649,589, which was a National Stagefiling under 35 U.S.C. 371 of International Application No.PCT/SE99/01671 filed Sep. 23, 1999 which was published in English onApr. 6, 2000 and claims benefit of Swedish Application Nos. 9803276-6and 9803710-4 filed respectively on Sep. 25, 1998 and Oct. 29, 1998.These applications are herein incorporated by reference in theirentirety for all purposes.

TECHNICAL FIELD

The present invention relates to a method for treating nerve disordersin a mammal or a vertebrate by administering at least two cytokineinhibitors, of which one is preferably a TNF inhibitor. The inventionalso relates to the use of at least two cytokine inhibitors, of whichone is preferably a TNF inhibitor in the preparation of pharmaceuticalcompositions for the treatment of nerve root injury.

The object of the present invention is to obtain an improved possibilityto treat nerve disorders, such as nerve root injury induced by discherniation, which may turn up e.g. as a radiating pain in the arm or leg(sciatica), as low back pain or as whiplash associated disorder, byblocking disk related cytokines.

BACKGROUND OF THE INVENTION

It is established that conditions such as sciatica and low back pain aredue to activation and irritation of intraspinal nervous structures bydisk derived substances (Olmarker K, Rydevik B, Nordborg C, Autologousnucleus pulposus induces neurophysiologic and histologic changes inporcine cauda equina nerve roots, Spine 1993; 18(11): 1425-32; OlmarkerK, Larsson K, Tumor necrosis factor alpha and nucleus-pulposus-inducednerve root injury, Spine 1998; 23(23): 2538-44; Olmarker K, Rydevik B,Selective inhibition of tumor necrosis factor-alpha prevents nucleuspulposus-induced thrombus formation, intraneural edema, and reduction ofnerve conduction velocity: possible implications for futurepharmacologic treatment strategies of sciatica, Spine 2001; 26(8):863-9). One key substance for inducing such irritation is TumorNecrosis Factor alpha (TNF or TNF-alpha). TNF is a proinflammatorycytokine that may sensitize a nerve root in a way that when it issimultaneously deformed mechanically, ectopic nerve may be elicitedlocally and interpreted by the brain as pain in the correspondingdermatome. TNF may also induce a nutritional deficit in the nerve rootby increasing the vascular permeability leading to intraneural edema,and by initiating intravascular coagulation by activation of adhesionmolecules at the surface of the endothelial cells (Olmarker K, RydevikB, Selective inhibition of tumor necrosis factor alpha prevents nucleuspulposus-induced thrombus formation, intraneural edema, and reduction ofnerve conduction velocity: possible implications for futurepharmacologic treatment strategies of sciatica, Spine 2001;26(8):863-9). Both these mechanisms may subsequently lead to a reduced bloodflow with a reduced supply of nutrients and elimination of metabolicwaist products. This reduction in nutrition may also induce sciatic painper se. TNF may also induce low back pain due to local irritation ofsensory nerve endings at the surface of the intervertebral disk. Thismay occur when the nucleus pulposus herniates out into the spinal canaland TNF produced and released from the disk cells may reach the nerveendings.

Disk herniation is a troublesome disorder, which can cause pronouncedpain and muscle dysfunction, and thereby loss of ability to work. Aherniation may occur in any disk in the spine but herniations in thelumbar and the cervical spine are most common. A disk herniation in thecervical spine may induce radiating pain and muscle dysfunction in thearm, which is generally referred to as cervical rhizopathy. Herniationin the lumbar spine may induce radiating pain and muscle dysfunction inthe leg. The radiating pain in the leg is generally referred to assciatica. Disk herniation will cause trouble to a varying degree, andthe pain may last for one or two months or in severe cases up to 6months. The arm or leg pain that can occur as a result of diskherniation can be very intense and may thus affect the individualpatient's whole life situation during the sickness period.

U.S. Pat. No. 5,703,092 discloses the use of hydroxamic acid compoundsand carbocyclic acids as metalloproteinase and TNF inhibitors, for thetreatment of arthritis and other related inflammatory diseases. No useof these compounds for the treatment of nerve root injuries is disclosedor suggested.

U.S. Pat. No. 4,925,833 discloses the use of tetracyclines to enhancebone protein synthesis and treatment of osteoporosis.

U.S. Pat. No. 4,666,897 discloses inhibition of mammalian collagenolyticenzymes by administering tetracyclines. The collagenolytic activity ismanifested by excessive bone resorption, periodontal disease, rheumatoidarthritis, ulceration of cornea, or resorption of skin or otherconnective tissue collagen.

However, neither this nor U.S. Pat. No. 4,925,833 disclose nerve rootinjury or the treatment thereof.

It has also been disclosed that selective inhibition may be efficient inreducing sciatic pain (Korhonen K, Karppinen J, Malmivaara A, Paimela L,Kyllonen E, Lindgren K-A, et al. Treatment of sciatica with infliximab,a monoclonal humanised chimaeric antibody against TNF. Trans.International Society for the Study of the Lumbar Spine 2002; Cleveland,Ohio, p. 14).

Low back pain affects approximately 80% of the population during theirlifetime in most countries. Except for being extremely common, it isalso one of the most costly disorders for the society. In Sweden alone,low back pain was estimated to cost $320,000,000 in 1997. The major partof the cost relates to indirect costs such as sick-compensation andreduced productivity, and only a minor part is related to direct costssuch as medical care and pharmacological substances.

In a minority of the cases (5%), there may be a known cause for the painsuch as intra spinal tumors, rheumatic diseases, infections and more. Inthese cases the treatment may be specifically aimed at the cause.However, in the majority of the cases of low back pain, the causeremains unknown. At present there is no direct way to treat low backpain with an unknown cause and existing treatment modalities only aim atsymptomatic relief.

Low Back Pain and Sciatica

It is necessary to make a distinction between low back pain and onespecific condition that is often linked to low back pain called“sciatica”. Sciatica refers to radiating pain into the leg according tothe dermatomal innervation area of a specific spinal nerve root. Thepain in sciatica is distinctly different from that of low back pain. Insciatica, the pain is sharp and intense, often described as“toothache-like”, and radiates down into the lower extremities, belowthe level of the knee. The experience of the pain is closely related tothe dermatomal innervation of one or more lumbar spinal nerve roots.Sciatica is also frequently related to neurological dysfunction in thatspecific nerve and may be seen as sensory dysfunction, reduced reflexesand reduced muscular strength. The sciatic pain thus seem to be aneuropathic pain, i.e. pain due to nerve injury, induced by sensitizedaxons in a spinal nerve root at the lumbar spinal level. The painexperienced by the patient at low back pain is more dull and isdiffusely located in the lower back. There is never any radiating paininto the leg.

Sciatica is the result of nerve injury, and the cause of sciatica has ananatomical correlate. Since 1934, sciatica is intimately linked to thepresence of a herniated intervertebral disc. However, although mostpatients with sciatica will display a herniated disc at radiologicalexamination, it is surprising that approximately 30% of an adultpopulation at the age of 40-50 years of age with no present or previoussciatica also have disc herniations when assessed by magnetic resonancetomography, so called “silent” disc herniations (Wiesel, Tsourmas et al.1984; Boden, Davis et al. 1990; Boos, Rieder et al. 1995; Boos, Dreieret al. 1997). The presence of silent disc herniations is intriguing tothe spine research community and seems to contradict the relationshipbetween disc herniations and sciatica.

Scientific Knowledge of the Pathophysiologic Mechanisms Behind Low BackPain

It is well known that the outer part of the annulus fibrosus of theintervertebral disc and the posterior longitudinal ligament areinnervated by C-fibers. Although there are no nerve fibers in the deeperpart of the annulus fibrosus or the nucleus pulposus in normal discs,nerves may reach these parts in degenerated discs through annular tears.

Silent Disc Herniations

As presented earlier, it is known that approximately one-third of anormal adult population who never suffered from sciatica haveradiological visible disc herniations. Since the presence of a discherniation is so intimately linked to the symptom of sciatica this issurprising, and at present there is no valid explanation for thisphenomenon.

However, “silent” in this regard only implies that the disc herniationsdid not produce sciatica. One may assume though that they produce othersymptoms.

Whiplash and Whiplash Associated Disorders (WAD)

About 10% to 20% of the occupants of a stricken vehicle in rear-end carcollisions suffer from whiplash injury. The injury may also occur as aresult of other types of accidents, such as train accidents, and suddenretardations. This injury is defined as a non-contactacceleration-deceleration injury to the head-neck system. It is mostoften caused by a rear-end car collision and there is no direct impacton the neck.

Presenting symptoms usually include neckpain, headaches, disequilibrium,blurred vision, parenthesize, changes in cognition, fatigue, insomniaand hypersensitivity to light and sound. Dizziness described in avariety of terms such as imbalance, light headedness

and vertigo also occur frequently and these symptoms may be associatedwith long-term disability.

Although neurologic and orthopedic examinations do not revealabnormalities in the majority of patients, the characteristics ofdizziness due to whiplash can be elucidated by means ofElectroNystagmoGraphic (ENG) evaluation. This examination is a methodthat is suitable for proving pathology in the oculo-vestibular system ofwhiplash-patients.

Until recently, the reason for the extent of injury was poorlyunderstood. In addition, due to the legal and insurance issues, theveracity of complaints of neck pain and other symptoms by people whosuffer from whiplash is commonly viewed as suspect.

Whiplash injuries can be quite complex and may include a variety ofrelated problems, such as joint dysfunction, and faulty movementpatterns, chronic pain and cognitive and higher center dysfunction.

When the cervical spine (neck) is subject to a whiplash injury, there isusually a combination of factors that contribute to the pain. Thesefactors must be addressed individually, while maintaining a “holistic”view of the patient.

The most significant factors may include one or more of the following:joint dysfunction, muscle dysfunction, and faulty movement patterns.

Joint Dysfunction

This occurs when one of the joints in the spine or limbs loses itsnormal joint play (resiliency and shock absorption). It is detectedthrough motion palpation, a procedure in which the doctor gently movesthe joint in different directions and assesses its joint play. When ajoint develops dysfunction, its normal range of movement may be affectedand it can become painful. In addition, joint dysfunction can lead to amuscle imbalance and muscle pain and a vicious cycle. The loss of jointplay can cause abnormal signals to the nervous system (there are anabundance of nerve receptors in the joint). The muscles related to thatjoint can subsequently become tense or, conversely, underactive. Theresulting muscle imbalance can place increased stress on the joint,aggravating the joint dysfunction that already exists.

Muscle Dysfunction

When joint dysfunction develops, muscles are affected. Some musclesrespond by becoming tense and overactive, while others respond bybecoming inhibited and underactive. In either case, these muscles candevelop trigger points. Trigger points are areas of congestion withinthe muscle where sensitizing compounds accumulate. These sensitizingcompounds can irritate the nerve endings within the muscle and producepain. This pain can occur in the muscle itself or can be referred pain(perceived in other areas of the body). Muscle related mechanisms mayalso give rise to abnormal signaling to the nervous system. This eventcan subsequently cause disruption of the ability of the nervous systemto properly regulate muscles in other parts of the body, leading to thedevelopment of faulty movement patterns.

Faulty Movement Patterns

It is thought that the intense barrage of pain signals from a traumaticinjury to the cervical spine can change the way the nervous systemcontrols the coordinated function of muscles. The disruption ofcoordinated, stable movement is known as faulty movement patterns.Faulty movement patterns cause increased strain in the muscles andjoints, leading to pain. They can involve the neck itself or can arisefrom dysfunction in other areas of the body such as the foot or pelvis.Instability is also considered part of faulty movement patterns. Thereare 2 types of instability that can occur in whiplash: passiveinstability—the ligaments of the neck are loosened, and dynamicinstability—the nervous system disruption causes a disturbance in thebody's natural muscular response to common, everyday forces. As a resultof instability, even mild, innocuous activities can become painful.

SUMMARY OF THE INVENTION

It has been found that the use of a TNF-alpha inhibitor, such as asubstance selected from the group consisting of metalloproteinaseinhibitors excluding methylprednisolone, tetracyclines includingchemically modified tetracyclines, quinolones, corticosteroids,thalidomide, lazaroids, pentoxifylline, hydroxamic acid derivatives,carbocyclic acids, napthopyrans, soluble cytokine receptors, monoclonalantibodies towards TNF-alpha, amrinone, pimobendan, vesnarinone,phosphodiesterase inhibitors, lactoferrin and lactoferrin derivedanalogs, and melatonin are suitable for treatment of spinal disordersand nerve root injury caused by the liberation of TNF-alpha andcompounds triggered by the liberation of or presence of TNF-alpha byinhibiting spinal disc TNF-alpha.

These substances are thus suitable for treatment of nerve root injury,and for treatment of sciatica, low back pain (LBP), and whiplashassociated disorder (WAD).

TNF is one of many pro-inflammatory substances with similar action, andit is considered as a “major player” in inflammatory events. However,TNF may also in part acts through other pro-inflammatory cytokines suchas for instance IL-1, IL-6, FAS, and IFN-gamma.

The present invention is based on the finding that by combining at leasttwo different inhibitors of pro-inflammatory cytokines it is possible toprovide an even better treatment of the above-mentioned diseases andconditions.

It is an object of the invention to provide novel and improved methodsfor inhibiting the action of cytokines for treating of nerve disordersin a subject comprising the step of administering to said subject atherapeutically effective dosage of at least two substances selectedfrom the group consisting of TNF inhibitors, IL-1 inhibitors, IL-6inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors, andIFN-gamma inhibitors.

A preferred embodiment of the invention is a method for treatment of anerve disorder in a subject comprising administering to a subject atherapeutically effective dosage of a TNF inhibitor in combination witha second inhibitor selected from the group consisting of IL-1inhibitors, IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligandinhibitors, and IFN-gamma inhibitors.

Another preferred embodiment of the invention is a method for treatmentof a nerve disorder in a subject comprising administering to a subject atherapeutically effective dosage of one TNF inhibitor, such as aspecific TNF inhibitor, in combination with another TNF inhibitor, suchas a non-specific TNF inhibitor.

Another preferred embodiment of the invention is a method for treatmentof a nerve disorder in a subject comprising administering to a subject atherapeutically effective dosage of a TNF inhibitor in combination witha IL-1 inhibitor.

It is also an object of the invention to provide a novel pharmaceuticalcomposition for treating nerve disorders in a subject comprising atherapeutically effective dosage of at least two substances selectedfrom the group consisting of INF inhibitors, IL-1 inhibitors, IL-6inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors, andIFN-gamma inhibitors.

Nerve disorders treatable with the method and the pharmaceuticalcomposition according to the invention are nerve disorders due to areduced nerve reduction velocity, spinal disorders, nerve root injuries,nerve disorders caused by disc herniation, sciatica, cervicalrhizopathy, low back pain, whiplash associated disorder, nerve disordersinvolving pain, nucleus pulposus-induced nerve injuries, and spinal cordcompressions.

The subject which can be treated by these methods include anyvertebrate, preferably mammals, and of those, most preferably humans.

Although a break-through in the treatment of spinal pain syndromes wasmade in 1997 when the involvement of pro-inflammatory cytokines, inparticular TNF, became evident, the current invention offers an evenmore efficient way to treat i.a. sciatica and low back pain bypharmacological means. Since TNF acts synergistically with otherpro-inflammatory cytokines inhibition of more cytokines than TNF is moreefficient in acquiring the desired clinical effect.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

It has now surprisingly been shown possible to be able to treat nerveroot injuries, or at least alleviate the symptoms of nerve root injuriesby using a pharmaceutical composition comprising a therapeuticallyactive amount of a TNF-alpha inhibitor. TNF-alpha inhibitors, includebut are not limited to, metalloproteinase (MMP) inhibitors (excludingmethylprednisolone), tetracyclines, chemically modified tetracyclines,quinolones, corticosteroids, thalidomide, lazaroids, pentoxifylline,hydroxamic acid derivatives, napthopyrans, soluble cytokine receptors,monoclonal antibodies towards TNF-alpha, amrinone, pimobendan,vesnarinone, phosphodiesterase inhibitors, lactoferrin and lactoferrinderived analogous, and melatonin in the form of bases or addition saltstogether with a pharmaceutically acceptable carrier.

By “therapeutically active amount” and “therapeutically effectivedosage” are intended to be an amount that will lead to a desiredtherapeutic effect, i.e., an amount that will lead to an improvement ofthe patient's condition. In one preferred example, an amount sufficientto ameliorate or treat a condition associated with a nerve disorder.

By “mammal” is meant to include but is not limited to primate, human,canine, porcine, equine, murine, feline, caprine, ovine, bovine, lupine,camelid, cervidae, rodent, avian and ichthyes. By animal is meant toinclude any vertebrate animal wherein there is a potential for nerveroot injury.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab fragments, scFv, and F(ab)₂ fragmentsthereof. Complete, intact antibodies include monoclonal antibodies suchas murine monoclonal antibodies (mAb), chimeric antibodies, humanizedantibodies and human. The production of antibodies and the proteinstructures of complete, intact antibodies, Fab fragments, scFv fragmentsand F(ab)₂fragments and the organization of the genetic sequences thatencode such molecules, are well known and are described, for example, inHarlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988) and Harlow et al., USINGANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press, 1999, whichare herein incorporated by reference in their entirety.

By “epitope” is meant a region on an antigen molecule to which anantibody or an immunogenic fragment thereof binds specifically. Theepitope can be a three dimensional epitope formed from residues ondifferent regions of a protein antigen molecule, which, in a naivestate, are closely apposed due to protein folding. “Epitope” as usedherein can also mean an epitope created by a peptide or hapten portionof TNF-alpha and not a three dimensional epitope. Preferred epitopes arethose wherein when bound to an immunogen (antibody, antibody fragment,or immunogenic fusion protein) results in inhibited or blocked TNF-alphaactivity.

By “TNF-alpha blocking” is meant a compound or composition that blocks,inhibits or prevents the activity of TNF or TNF-alpha.

Compounds that possess TNF-alpha inhibitory activity are for exampletetracyclines, (e.g., tetracycline, doxycycline, lymecycline,oxytetracycline, minocycline), and chemically modified tetracyclines(e.g., dedimethylamino-tetracycline), hydroxamic acid compounds,carbocyclic acids and derivatives, thalidomide, lazaroids,pentoxifylline, napthopyrans, soluble cytokine receptors, monoclonalantibodies towards INF-alpha, amrinone, pimobendan, vesnarinone,phosphodiesterase inhibitors, lactoferrin and lactoferrin derivedanalogs, melatonin, norfloxacine, ofloxacine, ciprofloxacine,gatifloxacine, pefloxacine, lomefloxacine, temafloxacine, TTP and p38kinase inhibitors. These compounds can be present as bases or in theform of addition salts, whichever possesses the best or preferredpharmaceutical effect, and best property to be brought into a suitablepharmaceutical composition. A more complete list is given below.

As stated above, there are several different types of cytokine blockingsubstances and pharmacological preparations that may be used accordingto the invention, and those substances may be grouped in differentsubclasses:

TNF inhibitors Specific TNF inhibitors Monoclonal antibodies such as:infliximab, CDP-571 (HUMICADE ™), D2E7 (Adalimumab), and CDP-870;Polyclonal antibodies; Soluble cytokine such as: receptors etanercept,lenercept, pegylated TNF receptor type 1, and TBP-1; TNF receptorantagonists; Antisense such as: oligonucleotides; ISIS-104838Non-specific TNF inhibitors 5,6-dimethylxanthenone- 4-acetic acid(acemannan); AGT-1; ANA 245; AWD 12281; BN 58705; Caspase inhibitors;CBP-1011; CC 1069; CC 1080; CDC 801; CDDO; CH-3697; CLX 1100; CM 101;CT3; CT 2576; CPH 82; CV 1013; Cyclosporin; Compounds used in anti- suchas: cancer treatment the binuclear DNA threading transition metalcomplexes and pharmaceutical compositions comprising them described inWO 99/15535, and methotrexate; Declopramide; DPC 333; DWP 205297; DY9973; Edodekin alfa; Flt ligand (available from Immunex); Galliumnitrate; HP 228; Hydroxamic acid derivates; IL-12; IL-18; Ilodekacin;Ilomastat; ITF-2357; JTE 607; Lactoferrin; Lactoferrin derived or suchas: derivable peptides the peptides described in WO 00/01730; Lazaroids;nonglucocorticoid 21- such as: aminosteroids U-74389G (16- desmethyltirilazad), and U- 74500; LPS agonist Esai; Melancortin agonists suchas: HP-228; Mercaptoethylguanidine; Metoclopramide: MMP inhibitors (i.e.matrix metalloproteinase inhibitors or TACE inhibitors, i.e. TNF AlphaConverting Enzyme- such as: inhibitors) Tetracyclines such as:Doxycycline, Lymecycline, Oxitetracycline, Tetracycline, andMinocycline; Synthetic tetracycline derivates (CMT Chemically ModifiedTetracyclines); KB-R7785; TIMP1 and TIMP2; adTIMP2 and adTIMP2; M-PGA;Napthopyrans: NCS-700; Nimesulide; NR58-3.14.3; p38 kinase inhibitorssuch as: VX-702, VX-740, VX-745 (Pralnacasan), VX-765, VX-850,SB-202190, SB-203580, and Pyridinyl imidazoles; PCM-4; PD-168787;Pentoxifyllin derviates; Pharma projects no. 6181, 6019 and 4657;Phosphodiesterase I, II, such as: III, IV, and V-inhibitors CC-1088, Ro20-1724, rolipram, amrinone, pimobendan, vesnarinone, and SB 207499;Piclamastat; PMS-601; Prostaglandins such as: Iloprost (prostacyclin);Quinolones (chinolones) such as: Norfloxacin, Levofloxacin, Enoxacin,Sparfloxacin, Temafloxacin, Moxifloxacin, Gatifloxacin, Gemifloxacin,Grepafloxacin, Trovafloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin,Lomefloxacin, and Temafloxacin; RDP-58; RIP-3; Sch-23863; SH-636;Solimastat; SR-31747; Tasonermin; Thalidomide derivates (or SelCID $$Selective Cytokin inhibitors, e.g. such as: thalidomide derivate)CC-1088 CDC-501, and CDC-801; TNF alpha proteinase inhibitor availablefrom Immunex; TNF-484A; Tristetraproline (TTP) (available fromAstraZeneca); VRCTC 310; Yissum project no. 11649; Zanamivir Inhibitorsof Interleukin-1 alpha and beta (IL-1α and IL-1β) Specific inhibitors ofIL-1α and IL-1β Monoclonal antibodies such as: CDP-484; Soluble cytokinereceptors; IL-1 type II receptor (decoy RII); Receptor antagonists suchas: IL-1ra, anakinra (KINERET ®), and ORTHOKIN ®; Antisenseoligonucleotides Non-specific inhibitors of IL-1α and IL-1β MMPinhibitors (i.e. such as: matrix Tetracyclines such as: Doxycycline,metalloproteinase Trovafloxacin, inhibitors), Lymecycline,Oxitetracycline, Tetracycline, Minocycline, and synthetic tetracyclinederivatives, such as CMT, i.e. Chemically Modified Tetracyclines;Prinomastat (AG3340); Batimastat; Marimastat; BB-3644; KB-R7785; TIMP-1,and TIMP-2, adTIMP-1 (adenoviral delivery of TIMP-1), and adTIMP-2(adenoviral delivery of TIMP-2); Quinolones (chinolones) such as:Norfloxacin, Levofloxacin, Enoxacin, Sparfloxacin, Temafloxacin,Moxifloxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Trovafloxacin,Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin, Temafloxacin;Prostaglandins: Iloprost (prostacyclin); Phosphodiesterase I, II, III,IV, and V-inhibitors; CC-1088, Ro 20-1724, rolipram, amrinone,pimobendan, vesnarinone, SB 207499 Inhibitors of Interleukin-6 (IL-6)Specific inhibitors of IL-6 Monoclonal antibodies; Soluble cytokinereceptors; Receptor antagonists; Antisense oligonucleotides Non-specificinhibitors of IL-6 MMP inhibitors (i.e. such as: matrix Tetracyclinessuch as: metalloproteinase Doxycycline, inhibitors) Lymecycline,Oxitetracycline, Tetracycline, Minocycline, and synthetic tetracyclinederivatives, such as CMT, i.e. Chemically Modified Tetracyclines;Prinomastat (AG3340); Batimastat; Marimastat; BB-3644; KB-R7785; TIMP-1,and TIMP-2, adTIMP-1 (adenoviral delivery of TIMP-1), and adTIMP-2(adenoviral delivery of TIMP-2); Quinolones (chinolones) such as:Norfloxacin, Levofloxacin, Enoxacin, Sparfloxacin, Temafloxacin,Moxifloxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Trovafloxacin,Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin, Temafloxacin;Prostaglandins; Iloprost (prostacyclin); Cyclosporin Pentoxifyllinderivates; Hydroxamic acid derivates; Phosphodiesterase I, II, III, IV,and V-inhibitors; CC-1088, Ro 20-1724, rolipram, amrinone, pimobendan,vesnarinone, SB 207499; Melanin and melancortin agonists; HP-228Inhibitors of Interleukin-8 (IL-8) Specific inhibitors of IL-8Monoclonal antibodies; Soluble cytokine receptors; Receptor antagonists;Antisense oligonucleotides; Non-specific inhibitors of IL-8 Quinolones(chinolones) such as: Norfloxacin, Levofloxacin, Enoxacin, Sparfloxacin,Temafloxacin, Moxifloxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin,Trovafloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin,Temafloxacin: Thalidomide derivates such as: SelCID. i.e. SelectiveCytokine inhibitors such as: CC-1088, CDC-501, CDC-801 and Linomide(Roquininex ®); Lazaroids; Cyclosporin; Pentoxifyllin derivates; FASInhibitors Specific FAS inhibitors Monoclonal antibodies: Solublecytokine receptors; Receptor antagonists; Antisense oligonucleotides;Non-specific FAS inhibitors Inhibitors of FAS ligands Specificinhibitors of FAS ligands Monoclonal antibodies; Soluble cytokinereceptors; Receptor antagonists; Antisense oligonucleotides;Non-specific inhibitors of FAS ligands Inhibitors of interferon-gamma(IFN-gamma) Specific IFN-gamma inhibitors Monoclonal antibodies; Solublecytokine receptors; Receptor antagonists; Antisense oligonucleotides;Non-specific IFN-gamma inhibitors MMP inhibitors (i.e. such as: matrixTetracyclines such as: metalloproteinase Doxycycline, inhibitors)Trovafloxacin, Lymecycline, Oxitetracycline, Tetracycline, Minocycline,and synthetic tetracycline derivatives, such as CMT, i.e. ChemicallyModified Tetracyclines; Prinomastat (AG3340); Batimastat; Marimastat;BB-3644; KB-R7785; TIMP-1, and TIMP-2, adTIMP-1 (adenoviral delivery ofTIMP-1), and adTIMP-2 (adenoviral delivery of TIMP-2); Quinolones(chinolones) such as: Norfloxacin, Levofloxacin, Enoxacin, Sparfloxacin,Temafloxacin, Moxifloxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin,Trovafloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin,Temafloxacin, Rebamipide, and Nalidixic acid; Lazaroids; Pentoxifyllinderivates; Phosphodiesterase I, II, III, IV, and V-inhibitors; CC-1088,Ro 20-1724, rolipram, amrinone, pimobendan, vesnarinone, SB 207499;Also contemplated are the pharmaceutically acceptable bases and salts ofthe substances listed above.

Preferred groups of TNF-alpha blocking substances for use according tothe present invention are soluble cytokine receptors, monoclonalantibodies, and tetracyclines or chemically modified tetracyclines.

Two preferred substances for use according to the present invention arethe monoclonal antibodies, D2E7 and CDP-870.

D2E7 is a fully humanized monoclonal antibody directed against humanTNF-alpha, which has been developed by Knoll and Cambridge AntibodyTechnology. A transgenic recombinant version of this antibody is underdevelopment by Genzyme Transgenic. The invention contemplates anyantibody that binds to the same epitope as D2E7 or that has the sameTNF-alpha inhibitory effect as D2E7. Preferably the antibody isprimatized®, humanized or human.

CDP-870 (or CDP 870) is a humanized antibody fragment with high affinityto TNF-alpha. It has been developed by Celltech Group plc, and isco-developed with Pharmacia Corporation. The invention contemplates anyantibody, antibody fragment or immunogen that binds to the same epitopeas CDP-870 or that has the same TNF-alpha inhibitory activity asCDP-870. Preferably the antibody, antibody fragment or immunogen has thesame or similar TNF-alpha inhibitory activity. Preferably the antibody,antibody fragment or immunogen is primatized, humanized or human.

According to a preferred embodiment, one of the substances used is a TNFinhibitor. According to a preferred variant of this embodiment the TNFinhibitor is a monoclonal antibody directed against TNF, such asinfliximab, CDP-571, D2E7 or CDP870. According to another preferredvariant of this embodiment the TNF inhibitor used is a soluble cytokineTNF receptor, such as etanercept. According to another preferred variantof this embodiment the TNF inhibitor used is a binuclear DNA threadingtransition metal complex with anti-cancer effect. According to anotherpreferred variant of this embodiment the TNF inhibitor used is alactoferrin derivable peptide. According to another preferred variant ofthis embodiment the TNF inhibitor used is an MMP inhibitor, such asdoxycycline. According to another preferred variant of this embodimentthe TNF inhibitor used is a p38 kinase inhibitor. According to yetanother preferred variant of this embodiment the substance used is TTP.

According to another preferred embodiment, one of the substances is aspecific TNF inhibitor, such as infliximab, CDP-571, D2E7 or CDP-870,which is use in combination with a non-specific TNF inhibitor, such asdoxycycline.

Doxycycline inhibits the action of TNF in a non-specific manner. TNF andother similar bioactive substances are first produced in an inactiveform and transported to the cell membrane. Upon activation, the activepart of the pro-TNF is cleaved and released. This process is calledshedding and may be initiated by one or more enzymes. These enzymes havein common that they are metalloproteinases, i.e. dependent of ametal-ion for their function. Doxycycline and other tetracyclines areknown to bind to metal-ions and will thereby inhibit the action ofmetalloproteinases and subsequently the release of TNF and otherpro-inflammatory cytokines in a non-specific manner. A monoclonalanti-TNF antibody, on the other hand, will bind directly to TNF andthereby inhibit TNF in a more specific way than doxycycline. Theinhibition may thus be assumed to be more efficient but will berestricted to TNF. However, in the work leading to the presentinvention, it was found that anti-TNF treatment was more efficient thandoxycycline treatment. However, the most efficient way to inhibit thenucleus pulpous induced reduction in nerve root conduction velocity wasfound to be to combine a TNF-inhibitor and an IL-1 inhibitor. TNF isknown to be orchestrating much of the inflammatory events. Except forhaving direct effects on target-receptors, it may also act through othercytokines by stimulation of their release. Although one may assume thatspecific inhibition may block both the direct effects of TNF and itsstimulatory effects on other cytokines, the work leading to the presentinvention showed that it is possible to further inhibit the action ofTNF by inhibiting cytokines that are produced and released by TNFstimulation. In addition to inhibiting the stimulatory effects of TNF,one thereby also inhibits the effects of these synergistic cytokinesdownstream. Since these synergistic cytokines may also induce releaseand production of TNF, the inhibition of these cytokines also reducesthe level of TNF. According to the present invention it is shown thatthe use of also one or more inhibitors of other cytokines is moreefficient in blocking the nucleus pulposus induced effects on adjacentnerve roots.

The combination of inhibitors of one specific cytokine with differentmechanisms is also shown to be more efficient than the use of a singleinhibitor. For instance, TNF may be inhibited at the synthesis-level(e.g., by pentoxifylline), at translation by antisense (e.g., byISIS-104838), at shedding (e.g., by doxycycline), and later byantibodies (e.g., by infliximab or CDP-870) or soluble receptors (e.g.,by etanercept or lenercept). Thus, there are at least five mechanismsuseful for inhibiting TNF. The combination of two or more drugs that actthrough different mechanisms therefore induces a more efficientinhibition of that certain cytokine than the use of one single drug. Aspecial benefit is achieved if a specific inhibitor or a key substance,for instance a monoclonal antibody against TNF, and a non-specificinhibitor that also blocks other cytokines, for instance doxycycline.This combination achieves inhibition of TNF at two levels (shedding andbinding of active TNF) as well as inhibition of other synergisticcytokines.

According to another preferred embodiment one of the substances used isan IL-1 inhibitor.

According to an especially preferred embodiment one of the substancesused is a TNF inhibitor and one is an IL-1 inhibitor.

Said at least two substances are preferably administered simultaneously,but they may also be administered separately.

The substances according to the invention may also be administered incombination with other drugs or compounds, provided that these otherdrugs or compounds do not eliminate the desired effects according to thepresent invention, i.e., the effect on TNF-alpha.

The invention further relates to a method for inhibiting the symptoms ofnerve root injury.

The effects of doxycycline, soluble cytokine-receptors, and monoclonalcytokineantibodies have been studied and representative methods used andresults obtained are disclosed below. Although the present invention hasbeen described in detail with reference to examples herein, it isunderstood that various modifications call be made without departingfrom the spirit of the invention, and would be readily known to theskilled artisan.

The compounds of the invention can be administered in a variety ofdosage forms, e.g., orally (per os), in the form of tablets, capsules,sugar or film coated tablets, liquid solutions; rectally, in the form ofsuppositories; parenterally, e.g., intramuscularly (i.m.), subcutaneous(s.c.), intracerebroventricular (i.c.v.), intrathecal (it.), epidurally,transepidermally or by intravenous (iv.) injection or infusion; byinhalation; or intranasally.

The therapeutic regimen for the different clinical syndromes may beadapted to the disease or condition, medical history of the subject aswould be know to the skilled artisan or clinician. Factors to beconsidered but not limiting to the route of administration, the form inwhich the compound is administered, the age, weight, sex, and conditionof the subject involved.

For example, the oral route is employed, in general, for all conditions,requiring such compounds. In emergency cases, preference is sometimesgiven to intravenous injection. For these purposes, the compounds of theinvention can be administered, for example, orally at doses ranging fromabout 20 to about 1500 mg/day. Of course, these dosage regimens may beadjusted to provide the optimal therapeutic response depending on thesubject's condition.

The nature of the pharmaceutical composition containing the compounds ofthe invention in association with pharmaceutically acceptable carriersor diluents will, of course, depend upon the desired route ofadministration. The composition may be formulated in the conventionalmanner with the usual ingredients. For example, the compounds of theinvention may be administered in the form of aqueous or oily solutionsor suspensions, tablets, pills, gelatin capsules (hard or soft ones),syrups, drops or suppositories.

For oral administration, the pharmaceutical compositions containing thecompounds of the invention are preferably tablets, pills or gelatinecapsules, which contain the active substance or substances together withdiluents, such as lactose, dextrose, sucrose, mannitol, sorbitol,cellulose; lubricants, e.g., silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; or they may also containbinders, such as starches, gelatine, methyl cellulose,carboxymethylcellulose, gum arabic, tragacanth, polyvinylpyrrolidone;disaggregating agents such as starches, alginic acid, alginates, sodiumstarch glycolate, microcrystalline cellulose; effervescing agents, sucha carbonates and acids; dyestuffs; sweeteners; wetting agents, such aslecithin, polysorbates, laurylsulphates; and in general non-toxic andpharmaceutically inert substances used in the formulation ofpharmaceutical compositions. Said pharmaceutical compositions may bemanufactured in known manners, e.g., by means of mixing, granulating,tableting, sugar-coating or film-coating processes. Film providingcompounds can be selected to provide release in the right place or atthe appropriate time in the intestinal tract with regard to absorptionand maximum effect. Thus pH-dependent film formers can be used to allowabsorption in the intestines as such, whereby different phthalates arenormally used or acrylic acid/methacrylic acid derivatives and polymers.

The liquid dispersions for oral administration may be, e.g., syrups,emulsions, and suspensions.

The syrups may contain as carrier, e.g., saccharose, or saccharose withglycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as Garner, e.g., a natural gum,such as gum arabic, xanthan gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, polyvinyl alcohol.

The suspension or solutions for intramuscular injections may containtogether with the active compound, a pharmaceutically acceptablecarrier, such as e.g., sterile water, olive oil (or other vegetable ornut derived oil), ethyl oleate, glycols“, e.g., propylene glycol, and ifso desired, a suitable amount of lidocaine hydrochloride. Adjuvants fortriggering the injection effect can be added as well.

The solutions for intravenous injection or infusion may contain ascarrier, e.g., sterile water, or preferably, a sterile isotonic salinesolution, as well as adjuvants used in the field of injection of activecompounds. Such solutions would also be suitable for i.m. and i.c.v.injection.

The suppositories may contain together with the active compounds, apharmaceutically acceptable carrier, e.g., cocoa-butter polyethyleneglycol, a polyethylene sorbitan fatty acid ester surfactant or lecithin.

Examples of suitable doses of the active agents contemplated fordifferent administration routes are given below.

1 Per os 10-300 mg i.m. 25-100 mg i.v. 2.5-25 mg i.t. 0.1-25 mg(daily—every 3.sup.rd month) inhalation 0.2-40 mg transepidermally10-100 mg intranasally 0.1-10 mg s.c. 5-10 mg i.c.v. 0.1-25 mg(daily—every 3.sup.rd month) epidurally 1-100 mg

These ranges are approximate (e.g., about 1 to about 100) and may varydepending on the specific agent being administered and the nature of thedisorder in the subject. Thus, it is further contemplated that anydosage in between for the cited ranges may also be used.

Examples of suitable doses for different TNF-alpha inhibitors are givenin the table below.

-   Preferred dosage of TNF-alpha blocking substance and administration    route-   Lenercept i.v. 5-200 10-100 30-80 (all given in mg for    administration once every 4th week)-   TBP-1 i.v. 5-200 10-100 30-80 (all given in mg for administration    once every 4th week)-   CDP-571 (HUMICADE) i.v. 1-100 5-10 5-10 (all given in mg/kg body    Weight for administration as a single dose)-   D2E7 i.v. 0.1-50 0.5-10 1-10 s.c. 0.1-50 0.5-10 1-10 (all given in    mg/kg body weight for administration as a single dose)-   Iloprost i.v. 0.1-2000 1-1500 100-1000 (all given in ug/kg body    weight/day) intranasally 50-250 100-150 100-150 (all given in    ug/day)-   Thalidomide 100-1200 300-1000 500-800 (all given in ug/day) CC-I088    Per os 50-1200 200-800 400-600 (all given in mg/day)-   CDP-870 i.v. 1-50 2-10 3-8 (all given in mg/kg body weight for    administration once every 4th week)-   HP-228 i.v. 5-100 10-50 20-40 (all given in .mu.g/kg body weight)-   ISIS-10483 Per os 1-100 10-50 20-50 s.c. 1-100 10-50 20-50 i.v.    1-100 10-50 20-50 (all given in mg)-   ARIFLO (SB 207499 Per os 10-100 30-60 30-45 (all given in mg/day)-   KB-R7785 s.c. 100-500 100-300 150-250 (all given in mg/kg body    weight/day)-   CDC-501 Per os 50-1200 200-800 400-600 (all given in mg/day)-   CDC-801 (ROQUININEX) Per os 50-1200 200-800 400-600 (all given in    mg/day)-   Prinomastat, Batimastat, and Marimastat Per os 1-250 mg 5-100 mg    10-50 mg (all given in mg twice/day)-   Linomide Per os 0.1-25 5-20 110-15 (all given in mg/kg body    weight/day)-   IL-1 blocking substance and administration route-   Anakinra (KINERET®) s.c. 10-200 50-150 100 (all given in mg/day)

Incorporation by Reference and Examples

Although the present invention has been described in detail withreference to examples below, it is understood that various modificationscan be made without departing from the spirit of the invention, andwould be readily known to the skilled artisan. All cited patents andpublications referred to in this application are herein incorporated byreference in their entirety for all purposes.

The application incorporates herein by reference in their entiretyInternational Application No. PCT/SE99/01670 and Swedish ApplicationNos. 9803276-6 and 9803710-4 for all purposes.

EXAMPLES Example 1

Study Design

The effects of nucleus pulposus and various treatments to blockTNF-activity were evaluated in an experimental set-up usingimmunohistochemistry and nerve conduction velocity recordings.

Summary of Background Data

A meta-analysis of observed effects induced by nucleus pulposus revealsthat these effects might relate to one specific cytokine, Tumor NecrosisFactor alpha (TNF-alpha). Objectives

To assess the presence of TNF-alpha in pig nucleus pulposus cells and tosee if blockage of TNF-alpha also blocks the nucleus pulposus-inducedreduction of nerve root conduction velocity.

Methods

Series-1: Cultured nucleus pulposus-cells were immunohistologicallystained with a monoclonal antibody for TNF:alpha.

Series-2: Nucleus pulposus was harvested from lumbar discs and appliedto the sacrococcygeal cauda equina in 13 pigs autologously. Four pigsreceived 100 mg of doxycycline intravenously, 8 pigs had a blockingmonoclonal antibody to TNF-alpha applied locally in the nucleuspulposus, and 4 pigs remained non-treated (controls). Three days afterthe application the nerve root conduction velocity was determined overthe application zone by local electrical stimulation.

Series-3: Thirteen pigs had autologous nucleus pulposus placed ontotheir sacrococcygeal cauda equina similar to series-2. Five pigs (bodyweight 25 kg) received REMICADE® (infliximab) 100 mg i.v.preoperatively, and 8 pigs received ENBREL® (etanercept) 12.5 mg s.c.preoperatively and additionally 12.5 mg s.c. three days after theoperation. Seven days after the nucleus pulposus-application the nerveroot conduction velocity was determined over the application zone bylocal electrical stimulation according to series-2.

Results

Series-1: TNF-alpha was found to be present in the nucleuspulposus-cells.

Series-2: The selective antibody to TNF-alpha limited the reduction ofnerve conduction velocity. However, treatment with doxycyclinesignificantly blocked the nucleus pulposus-induced reduction ofconduction velocity.

Series-3: Both drugs (infliximab, and etanercept) blocked the nucleuspulposus induced nerve injury efficiently. Normal average nerveconduction velocities were found after 15 treatment with both of thesetwo drugs.

CONCLUSION

For the first time a specific substance, Tumor Necrosis Factor-alpha(TNF-alpha.), has been linked to the nucleus pulposus-induced effects ofnerve roots after local application. Although the effects of thissubstance may be synergistic with other similar substances, the data ofthe present study may be of significant importance for the continuedunderstanding of nucleus pulposus' biologic activity, and might also beof potential use for future treatment strategies of sciatica and othernerve root injury conditions or related conditions.

After previously being considered as just a biologically inactive tissuecomponent compressing the spinal nerve root at disc herniation, thenucleus pulposus has recently been found to be highly active, inducingboth structural and functional changes in adjacent nerve roots whenapplied epidurally (Kayama S, Konno S, Olmarker K, Yabuki S, Kikuchi S,Incision of the anulus fibrosis induces nerve root morphologic,vascular, and functional changes. An experimental study, Spine 1996; 21:2539-43; Olmarker K, Brisby H, Yabuki S, Nordborg C, Rydevik B, Theeffects of normal, frozen, and hyaluronidase-digested nucleus pulposuson nerve root structure and function, Spine 1997; 22: 4715; discussion476; Olmarker K, Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8; Olmarker K, Nordborg C, Larsson K, Rydevik B,Ultrastructural changes in spinal nerve roots induced by autologousnucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, Rydevik B, NordborgC, Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18: 1425-32).It has thereby been established that autologous nucleus pulposus mayinduce axonal changes and a characteristic myelin injury (Kayama S,Konno S, Olmarker K, Yabuki S, Kikuchi S, Incision of the anulusfibrosis induces nerve root morphologic, vascular, and functionalchanges. An experimental study, Spine 1996; 21: 2539-43; Olmarker K,Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8; Olmarker K, Nordborg C, Larsson K, Rydevik B,Ultrastructural changes in spinal nerve roots induced by autologousnucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, Rydevik B, NordborgC, Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18: 1425-32.),increased vascular permeability (Byrod G, Otani K, Brisby H, Rydevik B,Olmarker K, Methylprednisolone reduces the early vascular permeabilityincrease in spinal nerve roots induced by epidural nucleus pulposusapplication, J Orthop Res 1987; 18: 6: 983-7), infra vascularcoagulation (Kayama S, Konno S, Olmarker K, Yabuki S, Kikuchi S,Incision of the anulus fibrosis induces nerve root morphologic,vascular, and functional changes. An experimental study, Spine 1996; 21:2539-43; Olmarker K, Blomquist J, Stromberg J, Nanmnark, D, Thomsen P,Rydevik B, Inflammatogenic properties of nucleus pulposus, Spine 1995;20: 665-9.), and that membrane-bound structure or substances of thenucleus pulposus-cells are responsible for these effects (Kayama S,Konno S, Olmarker K, Yabuki S, Kikuchi S, Incision of the anulusfibrosis induces nerve root morphologic, vascular, and functionalchanges. An experimental study, Spine 1996; 21: 2539-43; Olmarker K,Brisby H, Yabuki S, Nordborg C, Rydevik B, The effects of normal,frozen, and hyaluronidase-digested nucleus pulposus on nerve rootstructure and function, Spine 1997; 22: 4715; discussion 476.). Theeffects have also been found to be efficiently blocked bymethylprednisolone and cyclosporin A (Arai I, Konno S, Otani K, KikuchiS, Olmarker K, Cyclosporin A blocks the toxic effects of nucleuspulposus on spinal nerve roots, Submitted; 01 marker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8.).When critically looking at these data, one realizes that there is atleast one cytokine that relates to all of these effects, TNF-alpha.

To assess if TNF-alpha may be involved in the nucleus pulposus inducednerve root injury, the presence of TNF-alpha in nucleus pulposus-cellswas assessed and was studied if the nucleus pulposus-induced effectscould be blocked by doxycycline, a soluble TNF-receptor, and a selectivemonoclonal TNF-alpha antibody, the latter administered both locally inthe nucleus pulposus and systemically.

Example 2

Material and Methods

Series-1, Presence of TNF-Alpha in Pig Nucleus Pulposus-Cells: Nucleuspulposus (NP) from a total of 13 lumbar and thoracic discs were obtainedfrom 10 pigs, which were used for other purposes. NP was washed once inHam's F12 medium (Gibco BRL, Paisley, Scotland) and then centrifuged andsuspended in 5 ml of collagenase solution in Ham's F12 medium (0.8mg/ml, Sigma Chemical Co., St Louis, Mo., USA) for 40 minutes, at37.degree. C. in 25 cm tissue culture flasks. The separated NP-cellpellets were suspended in DMEM/FI2 1:1 medium (Gibco BRL, Paisley,Scotland) supplemented with 1% L-glutamine 200 mM (Gibco BRL, Paisley,Scotland), 50 mg/ml gentamycine sulphate (Gibco BRL, Paisley, Scotland)and 10% fetal calf serum (FCS), (Gibco BRL, Paisley, Scotland). Thecells were cultured at 37.degree. C. and 5% C02 in air for 3-4 weeks andthen cultured directly on tissue culture treated glass slides (BectonDickinson & Co Labware, Franklin Lakes, N.J., USA). After 5 days on theglass slides, the cells were fixed in situ by exposing the slides toacetone for 10 minutes. After blocking irrelevant antigens byapplication of 3% H202 (Sigma Chemical Co., St Louis, Mo., USA) for 30minutes and Horse Serum (ImmunoPure ABC, peroxidase mouse IgG stainingkit nr.32028, Pierce, Rockford, Ill.) for 20 minutes, the primaryantibody (Antipig TNF-alpha monoclonal purified antibody, Endogen,Cambridge, Mass., USA, Ordering Code MP-390) was applied over night at+40.degree C, diluted at 1:10, 1:20 and 1:40 dilutions. For control, BSA(bovine serum albumin, Intergen Co, New York, USA) suspended in PBS(phosphate buffered saline, Merck, Darmstadt, Germany) was applied inthe same fashion. The next day the cells were washed with 1% BSA in PBSand the secondary antibody (ImmunoPure ABC, peroxidase mouse IgGstaining kit Cat. Cat. #32028, Pierce, Rockford, Ill.) was applied for30 minutes. To enhance this reaction, the cells were exposed toAvidin-Biotin complex for an additional 30 minutes (ImmunoPure ABC,peroxidase mouse IgG staining kit Cat. #32028, Pierce, Rockford, Ill.).The cells were then exposed to 20 mg of DAB (3,3-diaminobenzidinetetrahydrochloride No. D5905, Sigma Chemical Co., St Louis, Mo., USA)and 0.033 ml of 3% H20 2in 10 ml of saline for 10 minutes. The cellswere washed in PBS, dehydrated in a series of ethanol, mounted andexamined by light microscopy by an unbiased observer for the presence ofa brown coloration indicating the presence of TNF-alpha.

Series-2, Neurophysiologic Evaluation:

Thirteen pigs (body weight 25-30 kg) received an intramuscular injectionof 20 mg/kg body weight of KETALAR™ (ketamine, 50 mg/ml, Parke-Davis,Moms Plains, N.J.) and an intravenous injection of 4 mg/kg body weightof HYPNODIL® (methomidate chloride, 50 mg/ml, AB Leo, Helsingborg,Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon, 2 mg/ml,Janssen Pharmaceutica, Beerse, Belgium). Anesthesia was maintained byadditional intravenous injections of 2 mg/kg body weight of HYPNODIL®and 0.05 mg/kg body weight of STRESNIL®. The pigs also received anintravenous injection of 0.1 mg/kg of STESOLID NOVUM® (Diazepam, Dumex,Helsingborg, Sweden) after surgery.

Nucleus pulposus was harvested from the 5.sup.th lumbar disc through aretro peritoneal approach (Olmarker K, Rydevik B, Nordborg C, Autologousnucleus pulposus induces neurophysiologic and histologic changes inporcine cauda equina nerve roots, Spine 1993; 18:1425-32.).Approximately 40 mg of the nucleus pulposus was applied to thesacrococcygeal cauda equina through a midline incision and laminectomyof the first coccygeal vertebra. Four pigs did not receive any treatment(no treatment). Four other pigs received an intravenous infusion of 100mg of doxycycline (Vibramycino, Pfizer Inc., New York, USA) in 100 ml ofsaline over 1 hour. In 5 pigs, the nucleus pulposus was mixed with 100ml of a 1.11 mg/mL suspension of the anti-TNF-alpha antibody used inseries 1, before application.

Three days after the application, the pigs were re-anesthetized by anintramuscular injection of 20 mg/kg body weight of KETALAR® and anintravenous injection of 35 mg/kg body weight 25 of PENTOTHAL®(Thiopental sodium, Abbott lab, Chicago, Ill.). The pigs were ventilatedon a respirator. Anesthesia was maintained by an intravenous bolusinjection of 100 mglkg body weight of Chloralose((a)-D(+)-gluco-chloralose, Merck, Darmstadt, Germany) and by acontinuous supply of 30 mg/kg/hour of Chloralose. A laminectomy from the4.sup.th sacral to the 3-.sup.rd coccygeal vertebra was performed. Thenerve roots were covered with SPONGOSTANE® (Ferrosan, Denmark). Localtissue temperature was continuously monitored and maintained at37.538.0. degree. C. by means of a heating lamp.

The cauda equina was stimulated by two E2 subdermal platinum needleelectrodes (Grass Instrument Co., Quincy, Mass.) which were connected toa Grass SD9 stimulator (Grass Instrument Co., Quincy, Mass.) and gentlyplaced intermittently on the cauda equina first 10 mm cranial and then10 mm caudal to the exposed area. To ensure that only impulses fromexposed nerve fibers were registered, the nerve root that exited fromthe spinal canal between the two stimulation sites were cut. Anelectromyogram (EMG) was registered by two subdermal platinum needleelectrodes which were placed into the paraspinal muscles in the tailapproximately 10 mm apart. This procedure is reproducible and representsa functional measurement of the motor nerve fibers of the cauda equinenerve roots. The EMG was visualized using a Macintosh IIci computerprovided with Superscope software and MacAdios II AID converter (GWInstruments, Sommerville, Mass.) together with a Grass P18 preamplifier(Grass Instrument Co., Quincy, Mass.). The separation distance betweenthe first peaks of the EMG from the two recordings was determined, andthe separation distance between the two stimulation sites on the caudaequina was measured with calipers. The nerve conduction velocity betweenthe two stimulation sites could thus be calculated from these twomeasurements.

The person performing the neurophysiologic analyses was unaware of theexperimental protocol for the individual animal. After finishing thecomplete study, the data were arranged in the three experimental groupsand statistical differences between the groups were assessed byStudent's t-test. The experimental protocol for this experiment wasapproved by the local animal research ethics committee.

Series-3:

Thirteen pigs had autologous nucleus pulposus placed onto theirsacrococcygeal cauda equina similar to series-2. Five pigs (bodyweight25 kg) received the human/murine monoclonal antibody, REMICADE®(infliximab, Immunex Corporation, Seattle, Wash. 98101, USA) 100 mg i.v.preoperatively, and 8 pigs received ENBREL® (etanercept, Centocor B.V.,Leiden, the Netherlands) 12.5 mg s.c. preoperatively and additionally12.5 mg s.c. three days after the operation. Seven days after thenucleus pulposus-application the nerve root conduction velocity wasdetermined over the application zone by local electrical stimulationaccording to series-2. To blind the study, the neurophysiologicalevaluation was conducted in parallel to another study and the personperforming the analyses did not know from which study and what treatmenteach specific animal was subjected to. No non-treated animals wereincluded in the series-3 due to the pre-existing knowledge of nerveconduction velocity after seven days of either nucleus pulposus or fat(control) application. The statistical difference between the groups,infliximab, and etanercept, nucleus pulposus without treatment (positivecontrol from previous data) and application of retroperitoneal fat(negative control from previous data) was assessed by using ANOVA andFisher's PLSD at 5%.

Results

Series-I, Presence of TNF-Alpha in Pig Nucleus Pulposus-Cells:

Examples of the light microscopic appearance of the stained glassslides. In the sections using BSA in PBS as “primary antibody”(control), no staining was observed, ensuring that there was no labelingand visualization of irrelevant antigens. When the anti-TNF-alphaantibody was applied at 1:40 dilution there was only weak staining.However, the staining increased with diminishing dilutions of theantibody. The staining was seen in the soma of the cells, and it was notpossible to differentiate whether TNFalpha was located in the cytoplasm,on the cell surface bound to the cell-membrane, or both.

Series-2 Neurophysiologic Evaluation:

Application of non-modified nucleus pulposus and without any treatmentinduced a reduction in nerve conduction velocity similar to previousstudies (Table 1). In contrast, treatment with doxycycline completelyblocked this reduction (p<0.01 Student's t-test). Local application ofanti-TNF-alpha-antibody also induced a partial block of this reduction,although not as complete as doxycycline and was not statisticallysignificant as compared to the no treatment-series.

Series-3:

Treatment with both drugs seemed to prevent the nucleus pulposus-inducedreduction of nerve root conduction velocities, since the average nerveconduction velocity for both these treatment groups were close to theaverage conduction of the fat application series, as seen in a previousstudy (Table 2). The average nerve conduction velocity in pigs treatedwith ENBREL™ was statistically different from the average nerveconduction velocity in the series with pigs with no treatment. Theaverage new conduction velocity in the group treated with REMICADE™ wasalso statistically significantly different from the average nerveconduction velocity in the group with no treatment.

TABLE 1 Series 2 Treatment n NCV (m/s +/− SD) Local anti-TNF alpha 5 64+/− 28 Doxycycline 4 76 +/− 9  No treatment 4 46 +/− 12

TABLE 2 Series 3 Treatment n NCV (m/s +/− SD) Fat 5 76 +/− 11 ENBREL ® 878 +/− 14 REMICADE ® 5 79 +/− 15 No treatment 5 45 +/− 19

Data included from Olmarker K, Rydevik B, Nordborg C, Autologous nucleuspulposus induces neurophysiologic and histologic changes in porcinecauda equina nerve roots, Spine 1993; 18:1425-32.

DISCUSSION

The data of the present study demonstrated that TNF-alpha may be foundin nucleus pulposus-cells of the pig. If TNF-alpha was blocked by alocally applied selective monoclonal antibody, the nucleuspulposus-induced reduction of nerve root conduction velocity waspartially blocked, although not statistically significant as compared tothe series with non-treated animals. However, if animals were treatedsystemically with doxycycline, infliximab, and etanercept to inhibitTNF-alpha, the reduction of nerve conduction velocity was significantlyprevented.

In recent years, it has been verified that local application ofautologous nucleus pulposus may injure the adjacent nerve roots. Thus,it has become evident that the nerve root injury seen as disc herniationmay not be solely based on mechanical deformation of the nerve root, butmay also be induced by unknown “biochemical effects” related to theepidural presence of herniated nucleus pulposus. Although this newresearch field has generated many experimental studies, the mechanismsand substances involved are not fully known. It has been seen that localapplication of autologous nucleus pulposus may induce axonal injury(Kayama S, Konno S, Olmarker K, Yabuki S, Kikuchi S, Incision of theanulus fibrosis induces nerve root morphologic, vascular, and functionalchanges. An experimental study, Spine 1996; 21: 2539-43; Olmarker K,Brisby H, Yabuki S, Nordborg C, Rydevik B, The effects of normal,frozen, and hyaluronidase-digested nucleus pulposus on nerve rootstructure and function, Spine 1997; 22: 4715; discussion 476; OlmarkerK, Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8; Olmarker K, Myers R R, Pathogenesis of sciatic pain:Role of herniated nucleus pulposus and deformation of spinal nerve rootand DRG, Pain, 1998,78: 9-105; Olmarker K, Nordborg C, Larsson K,Rydevik B, Ultrastructural changes in spinal nerve roots induced byautologous nucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, RydevikB, Nordborg C, Autologous nucleus pulposus induces neurophysiologic andhistologic changes in porcine cauda equina nerve roots, Spine 1993; 18:1425-32), a characteristic injury of the myelin sheath (Kayama S, KonnoS, Olmarker K, Yabuki S, Kikuchi S, Incision of the anulus fibrosisinduces nerve root morphologic, vascular, and functional changes. Anexperimental study, Spine 1996; 21: 2539-43; Olmarker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8;Olmarker K, Myers R R, Pathogenesis of sciatic pain: Role of herniatednucleus pulposus and deformation of spinal nerve root and DRG, Pain,1998, 78: 9-105; Olmarker K, Nordborg C, Larsson K, Rydevik B,Ultrastructural changes in spinal nerve roots induced by autologousnucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, Rydevik B, NordborgC, Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18: 1425-32), alocal increase of vascular permeability (Byrod G, Otani K, Brisby H,Rydevik B, Olmarker K, Methylprednisolone reduces the early vascularpermeability increase in spinal nerve roots induced by epidural nucleuspulposus application, 1. Orthop. Res. 1987; 186: 983-7; Olmarker K,Blomquist J, Stromberg J, Nanmnark, U, Thomsen P, Rydevik B,Inflammatogenic properties of nucleus pulposus, Spine 1995; 20: 665-9)infra vascular coagulations, reduction of infra neural blood flow (OtaniK, Arai I, Mao G P, Konno S, Olmarker K, Kikuchi S, Nucleuspulposus-induced nerve root injury. The relationship between blood flowand nerve conduction velocity, Neurosurgery 1999; 45: 619-20), andleukotaxis (Olmarker K, Blomquist J, Stromberg J, Nanmnark, U, ThomsenP, Rydevik B, Inflammatogenic properties of nucleus pulposus, Spine1995; 20: 665-9). It has been seen that the nucleus pulposus-relatedeffects may be blocked efficiently by methylprednisolone (Olmarker K,Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8) and cyclosporin A (Arai I, Konno S, Otani K, KikuchiS, Olmarker K, Cyclosporin A blocks the toxic effects of nucleuspulposus on spinal nerve roots, Submitted, and slightly less efficientlyby indomethacin (Arai I, Mao G P, Otani K, Komo S, Kikuchi S, OlmarkerK, Indomethacin blocks nucleus pulposus related effects in adjacentnerve roots, Accepted for publication in Eura Spine J.), and lidocaine(Yabuki S, Kawaguchi Y, Olmarker K, Rydevik B, Effects of lidocaine onnucleus pulposus-induced nerve root injury, Spine, 1998; 23: 29:2383-89). Further, it has been understood that the effects are mediatedby the nucleus pulposus-cells (37), particularly by substances orstructures bound to the cell membranes (Kayama S, Olmarker K, Larsson K,Sjoren-Jansson E, Lindahl A, Rydevik D, Cultured, autologous nucleuspulposus cells induce structural and functional changes in spinal nerveroots, Spine, 1998; 23: 2155-8). When critically considering these data,it becomes evident that at least one specific cytokine could be relatedto these observed effects, Tumor Necrosis Factor-alpha (TNF-alpha).TNF-alpha may induce nerve injury (Liberski P P, Yanagihara R, NerurkarV, Gajdusek D C, Further ultrastructural studies of lesions induced inthe optic nerve by tumor necrosis factor alpha (TNF-alpha): a comparisonwith experimental Creutzfeldt-Jakob disease, Acta Neurobiol En (Warsz)1994; 54: 209-18; Madigan M C, Sadun A A, Rao N S, Dugel P D, Tenhula WN, Gill P S, Tumor necrosis factor-alpha (TNF-alpha)-induced opticneuropathy in rabbits, Neurol Res 1996; 18: 176-84; Petrovich M S, Hsu HY, Gu X, Dugal P, Heller K B, Sadun A A, Pentoxifylline suppression ofTNF-alpha mediated axonal degeneration in the rabbit optic nerve, NeurolRes 1997; 19: 551-4; Said G, Hontebeyrie-Joskowicz M, Nerve lesionsinduced by macrophage activation, Res Immunol 1992; 143: 589-99; WagnerR, Myers R R, Endoneurial injection of TNF-alpha produces neuropathicpain behaviours, Neuroreport 1996; 7: 2897-901), mainly seen as acharacteristic myelin injury that closely resembles the nucleuspulposus-induced myelin-injury (Liberski P P, Yanagihara R, Nerurkar V,Gajdusek DC, Further ultrastructural studies of lesions induced in theoptic nerve by tumor necrosis factor alpha (TNF-alpha): a comparisonwith experimental Creutzfeldt-Jakob disease, Acta Neurobiol En (Warsz)1994; 54: 209-18; Redford E J, Hall S M, Smith K J, Vascular changes anddemyelination induced by the intraneural injection of tumour necrosisfactor, Brain 1995; 118: 869-78; Sehnaj K W, Raine C S, Tumor necrosisfactor mediates myelin and oligodendrocytc damage in vitro, Ann Neurol1988; 23: 339-46; Sharief M K, Ingram D A, Swash M, Circulating tumornecrosis factor-alpha correlates with electrodiagnostic abnormalities inGuillain-Barre syndrome, Ann Neuro 11997; 42: 6873; Tsukamoto T,Ishikawa M, Yamamoto T, Suppressive effects of TNF-alpha on myelinformation in vitro, Acta Neurol Scand 1995; 91: 71-5; Villarroya H,Violleau K, Ben Younes-Chennoufi A, Baumann N, Myelin-inducedexperimental allergic encephalomyelitis in Lewis rats: tumor necrosisfactor alpha levels in serum of cerebrospinal fluid immunohistochemicalexpression in glial cells and neurophages of optic nerve and spinalcord, J Neuroimmunol 1996; 64: 55-61; Wagner R, Myers R R, Endoneurialinjection of TNF-alpha produces neuropathic pain behaviours, Neuroreport1996; 7: 2897-901; Zhu J, Bai X F, Mix E, Link H, Cytokine dichotomy inperipheral nervous system influences the outcome of experimentalallergic neuritis: dynamics of MRNA expression for IL-1 beta, IL-6,IL-10, IL-12, TNF-alpha, TNF-beta, and cytolysin, Clin ImmunolImmunopathol 1997; 84: 85-94). TNF-alpha may also induce an increase invascular permeability (Redford E J, Hall S M, Smith K J, Vascularchances and demyelination induced by the intra neural injection oftumour necrosis factor, Brain 1995; 118: 869-78; Wagner R, Myers R R,Endoneurial injection of TNFalpha produces neuropathic pain behaviours,Neuroreport 1996; 7: 2897-901) and initiate coagulation (Jurd K M,Stephens C J, Black M M, Hunt B J, Endothelial cell activation incutaneous vasculitis, Clin Exp Dermatol 1996; 21: 28-32; Nawroth P,Handley D, Matsueda G, De Waal R, Gerlach H, Blohm D, Stem D, Tumornecrosis factor/cachectin induced intra vascular fibrin formation inmeth A fibrosarcomas, J Exp Med 1988; 168: 637-47; van der Poll T,Jansen P M, Van Zee K J, Welborn M Br, de Jong I, Hack C E, Loetscher H,Lesslauer W, Lowry S F, Moidawer L L, Tumor necrosis factor-alphainduces activation of coagulation and fibrinolysis in baboons through anexelusive effect on the p55 receptor, Blood 1996; 88: 922-7). Further,TNF-alpha may be blocked by steroids (Baumgartner R A, Deramo V A,Beaven M A, Constitutive and inducible mechanisms for synthesis andrelease of cytokines in immune cell lines, J Immunol 1996; 157: 4087-93;Brattsand R, Linden M, Cytokine modulation by glucocorticoids:mechanisms and actions in cellular studies, Aliment Pharmacol Ther 1996;10: 81-90; Iwamoto S, Takeda K, Possible cytotoxic mechanisms of TNF invitro, Hum Cell 1990; 3: 107-12; Teoh K H, Bradley C A, Galt J, BurrowsH, Steroid inhibition of cytokine mediated vasodilation after wann heartsurgery, Circulation 1995; 92: 11347-53; Wershil B K, Furuta G T,Lavigne J A, Choudhury A R, Wang Z S, Galli S J, Dexamethasonecyclosporin A suppress mast cell-leukocyte cytokine cascades by multiplemechanisms, In Arch Allergy Immunol 1995; 107: 323-4.), and cyclosporinA (Dawson J, Hurtenbach U, MacKenzie A, Cyclosporin A inhibits the invivo production of interleukin-Ibeta and tumour necrosis factor alpha,but not interleukin-6, by a T-cell independent mechanism, Cytokine 1996;8: 882-8; Smith C S, Ortega G, Parker L, Shearer W T, Cyclosporin Ablocks induction of tumor necrosis factor-alpha in human B lymphocytes,Biochem Biophys Res Commun 1994; 204: 383-90; Wasaki S, Sakaida I,Uchida K, Kiinura T, Kayano K, Oldta K, Preventive effect of cyclosporinA on experimentally induced acute liver injury in rats, Liver 1997; 17:107-14; Wershil B K, Furuta G T, Lavigne J A, Choudhury A R, Wang Z S,Galli S J, Dexamethasone cyclosporin A suppress mast cell leukocytecytokine cascades by multiple mechanisms, Int Arch Allergy Immunol 1995;107: 323-4). However, the blocking effect on TNF-alpha is not sopronounced by NSAID (Garcia-Vicuna R, Diaz-Gonzalez F, Gonzalez-Alvaro1, del Pozo Ma, Mollinedo F, Cabanas C, Gonzalez-Amaro R, Sanchez-MadridF, Prevention of cytokine-induced changes in leucocyte adhesionreceptors by nonsteroidal antiinflammatory drugs from the oxicam family,Arthritis Rheum 1997; 40: 143-53; Gonzalez E, de la Cruz C, de NicolasR, Egido J, Herrero-Beaumont G, Long-term effect of nonsteroidalanti-inflammatory drugs on the production of cytokines and otherinflammatory mediators by blood cells of patients with osteosis, AgentsActions 1994; 41: 171-8; Herman J H, Sowder W G, Hess E V, Nonsteroidalantiflammatory drug modulation of prosthesis ppseudomembrane inducedbone resorption, J Rheunutol 1994; 21: 338-43) and very low or theagonized by lidocaine (Bidani A, Heming T A, Effects of lidocaine oncytosolic pH regulation and stimulas-induced effector functions inalveolar macrophages, Lung 1997; 175:349-61; Matsumori A, Ono K, NishioR, Nose Y, Sasayaina S, Amiodarone inhibits production of tumor necrosisfactor-alpha by human mononuclear cells: a possible mechanism for itseffect in heart failure, Circulation 1997; 96: 1386-9; Pichler W J,Zanni M, von Greyerz S, Schnyder B, Mauri-Hellweg D, Wendland, T, HighIL-5 production by human drug-specific T cell clones, Int Arch AllergyImmunol 1997; 113: 177-80; Takao Y, Mikawa K, Nishina Y, Maekawa N,Obara H, Lidocaine attenuates hyperoxic lung injury in rabbits, ActaAnaesthesiol Scand 1996; 40: 318-25).

It was recently observed that local application of nucleus pulposus mayinduce pain-related behavior in rats, particularly thermal hyperalgesia(Kawakami M, Tamaki T, Weinstein J N, Hashizume H, Nishi H, Meller S T,Pathomechanism of pain-related behaviour produced by allografts ofintervertebral disc in the rat, Spine 1996; 21: 2101-7; Olmarker K,Myers R R, Pathogenesis of sciatic pain: Role of herniated nucleuspulposus and deformation of spinal nerve root and DRG, Pain, 1998, 78:9-105). TNF-alpha has also been found to be related to suchpain-behavioristic changes (DeLeo J A, Colburn R W, Rickman A J,Cytokine and growth factor immunohistochemical spinal profiles in twoanimal models of mononeuropathy, Brain Res 1997; 759: 50-7, Oka T,Wakugawa Y, Hosoi M, Oka K, Hari T, Intracerebroventricular injection oftumor necrosis factor-alpha induces thermal hyperalgesia in rats,Neuroimmunomodulation 1996; 3: 135-40; Sommer C, Schmidt C, George A,Toyka K V, A metalloprotease-inhibitor reduces pain associated behaviourin mice with experimental neuropathy, Neurosci Lett 1997; 237:458;Wagner R, Myers R R, Endoneurial injection of TNF-alpha producesneuropathic pain behaviours, Neuroreport 1996; 7: 2897-901), and also toneuropathies in general (Lin X H, Kashima Y, Khan M, Heller K B, Gu X Z,Sadun A A, An immunohistochemical study of TNF-alpha in optic nervesfrom AIDS patients, Curr Eye Res 1997; 16: 1064-8; Sharief M K, Ingram DA, Swash M, Circulating tumor necrosis factor-alpha correlates withelectrodiagnostic abnormalities in Guillain-Barre syndrome, Ann Neurol1997; 42: 6873; Sommer C, Schmidt C, George A, Toyka K V, Ametalloprotease-inhibitor reduces pain associated behaviour in mice withexperimental neuropathy, Neurosci Lett 1997; 237:45-8; Sorkin L S, XiaoW H, Wagner R, Myers R R, Tumour necrosis factor-alpha induces ectopicactivity in nociceptive primary afferent fibres, Neuroscience 1997; 81:255-62). However there are no studies that have assessed the possiblepresence of TNFalpha in the cells of the nucleus pulposus.

To assess if TNF-alpha could be related to the observed nucleus pulposusinduced reduction in nerve root conduction velocity it was necessaryfirst to analyze if there was TNF-alpha in the nucleus pulposus-cells.The data clearly demonstrated that TNF-alpha was present in these cells.TNF-alpha is produced as a precursor (pro-TNF) that is bound to themembrane, and it is activated by cleavage from the cell-membrane by azinc-dependent metallo-endopeptidase (i.e., TNF-alpha converting enzyme,TACE) (Black R A, Rauch C T, Kozlosky C J, Peschon I J, Slack J L,Wolfson M F, Castner B J, Stocking K L, Reddy P, Srinivasan S, Nelson N,Boiani N, Schooley K A, Gerhart M, Davis R, Fitzner J N, Johnson R S,Paxton R J, March C J, Cerretti D P, A metalloproteinase disintegrinthat releases tumour-necrosis factor-alpha from cells, Nature 1997; 385:729-33; Gearing A J, Beckett P, Christodoulou M, Churchill M, ClementsJ, Davidson A H, Drummond A H, Galloway W A, Gilbert R, Gordon J L, etal., Processing of tumour necrosis factor-alpha precursor bymetalloproteinases, Nature 1994; 370: 555-7; Gazelle E J, Banda M J,Leppert D, Matrix metallo-proteinases in immunity, J Immunol 1996; 156:14; Robache-Gallea S, Bruneau J M, Robbe H, Morand V, Capdevila C,Bhatnagar N, Chouaib S, Roman-Roman S, Partial purification andcharacterization of a tumor necrosis factor-alpha converting activity,Eur J Immunol 1997; 27: 1275-82; Rosendahl M S, Ko S C, Long D L, BrewerM T, Rosenzweig D, Hedl E, Anderson L, Pyle S M, Moreland J, Meyers M A,Kohno T, Lyons D, Lichenstein H S, Identification and characterizationof a pro-tumor necrosis factor-alpha-processing enzyme from the ADAMfamily of zinc metalloproteases, J Biol Chem 1997; 272: 24588-93). Thismay thus relate well to experimental findings, where application of onlythe cell-membranes of autologous nucleus pulposus-cells induced nerveconduction velocity reduction, which indicated that the effects weremediated by a membrane-bound substance. Second, the effects of theTNF-alpha had to be blocked in a controlled manner. We then first choseto add the same selective antibody that was used forimmunohistochemistry in series 1, which is known to also block theeffects of TNF-alpha, to the nucleus pulposus before application. Also,we chose to treat the pigs with doxycycline, which is known to blockTNF-alpha (Kloppenburg M, B-an BM, de Rooij-Dijk H H, Mitenburg A M,Daha M R, Breedveld F C, Dijkmans B A, Verweij C, The tetracyclinederivative minocycline differentially affects cytokine production bymonocytes and T lymphocytes, Antimicrob Agents Chemother 1996; 40:934-40; Kloppenburg M, Verweij C L, Miltenburg A M, Verhoeven A J, DahaM R, Dikmans B A, Breeveld F C, The influence of tetracyclines on T cellactivation, Clin Exp. Immunol 1995; 102: 635-41; Milano S, Arcoleo F,D'Agostino P, Cillari E, Intraperitoneal injection of tetracyclinesprotects mice from lethal endotoxemia downregulating inducible nitricoxide synthase in various organs and cytokine and nitrate secretion inblood, Antimicrob Agents Chemother 1997; 41: 117-21; Shapira L, Houri Y,Barak V, Halabi A, Soskoine W A, Stabholz A, Human monocyte response tocementum extracts from periodontally diseased teeth: effect ofconditioning with tetracycline, J Periodontol 1996; 67: 682-7; ShapiraL, Houri Y, Barak V, Soskolne W A, Halabi A, Stabholz A, Tetracyclineinhibits Porphyromonas gingivalis lipopolysaccharide-induced lesions invivo and TNF processing in vitro, J Periodontal Res 1997; 32: 183-8).However, due to the low pH of the doxycycline preparation, it was chosento treat the pigs by intravenous injection instead of local addition tothe nucleus pulposus since nucleus pulposus at a low pH has been foundto potentiate the effects of the nucleus pulposus (Olmarker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8;Iwabuchi M, Rydevik B, Kikuchi S, Olmarker K, Methylprednisolone reducesthe early vascular permeability increase in spinal nerves by epiduralnucleus pulposus application, Accepted for publication in Spine).

Two recently developed drugs for specific TNF-alpha inhibition were alsoincluded in the study. Infliximab is a chimeric monoclonal antibodycomposed of human constant and murine variable regions. Infliximab bindsspecifically to human TNF-alpha. As opposed to the monoclonal antibodyused in series-2 for the 3-day observation period, infliximab was notadministered locally in the autotransplanted nucleus pulposus, butinstead was administered systemically in a clinically recommended dose(4 mg/kg).

Etanercept is a dimeric fusion protein consisting of the Fc portion ofhuman IgG. The drug, etanercept, was administered in a dosage comparableto the recommended dose for pediatric use (0.5 mg/kg, twice a week).

The data regarding nerve conduction velocity showed that the reductionwas completely blocked by the systemic-treatment and that the nerveconduction velocities in these series were close to the conductionvelocity after application of a control substance (retro peritoneal fat)from a previous study (Olmarker K, Rydevik B, Nordborg C, Autologousnucleus pulposus induces neurophysiologic and histologic changes inporcine cauda equina nerve roots, Spine 1993; 18: 1425-32). Applicationof the anti-TNF-alpha antibody to the nucleus pulposus also partiallyprevented the reduction in nerve conduction velocity. However, thereduction was not as pronounced as that observed for doxycycline, andthe velocity in this series was not statistically different to thevelocity in the series with untreated animals, given the wide deviationof the data.

The local anti-TNF-alpha antibody treatment only partially blocked thenucleus pulposus-induced reduction of nerve conduction velocity and thehigh standard deviation of the data could probably have at least threedifferent explanations. First, if looking at the specific data withinthis group, it was found that the nerve conduction velocity was low in 2animals (mean 37.5 m/s) and high in 3 animals (mean 81.3 m/s). There arethus 2 groups of distinctly different data within the anti-TNF-alphatreatment series. This will account for the high standard deviation andmight imply that the blocking effect was sufficient in 3 animals andinsufficient in 2 animals. The lack of effects in these animals could bebased simply on the amount of antibodies in relation to TNF-alphamolecules not being sufficient, and if a higher dose of the antibody hadbeen used, the TNF-alpha effects would thus have been blocked even inthese animals. Such a scenario could then theoretically imply thatTNF-alpha alone is responsible for the observed nucleus pulposus-inducedeffects, and that this could not be verified experimentally due to theamount of antibody being too low.

TNF-alpha may have various pathophysiologic effects. It may have directeffects on tissues such as nerve tissue and blood vessels, it maytrigger other cells to produce other pathogenic substances and it maytrigger release of more TNF-alpha both by inflammatory cells and also bySchwann-cells locally in the nerve tissue (Wagner R, Myers R R, Schwanncells produce tumor necrosis factor alpha: expression in injurednon-injured nerves, Neuroscience 1996; 73: 625-9). There is thus reasonto believe that even low amounts of TNF-alpha may be sufficient toinitiate these processes and that there is a local recruitment ofcytokine producing cells and a subsequent increase in production andrelease of other cytokines as well as TNF-alpha. TNF-alpha may thereforeact as the “ignition key” of the pathophysiologic processes and play animportant role for the initiation of the pathophysiologic cascade behindthe nucleus pulposus-induced nerve injury. However, the majorcontribution of TNF-alpha may be derived from recruited, aggregated andmaybe even extravasated leukocytes, and that successful pharmacologicblock ma_(y) be achieved only by systemic treatment.

In conclusion, for the first time a specific substance (TNF-alpha) hasbeen linked to the nucleus pulposus-induced nerve root injury. This newinformation may be of significant importance for the continuedunderstanding of nucleus pulposus-induced nerve injury as well asraising the question of the potential future clinical use ofpharmacological interference with TNF-alpha and related substances, fortreatment of sciatica.

The presence of TNF-alpha in pig nucleus pulposus-cells was thusimmunohistochemicall y verified. Block of TNF-alpha by a locally appliedmonoclonal antibody limited the nucleus pulposus-induced reduction ofnerve root conduction velocity, whereas intravenous treatment withdoxycycline, infliximab, and etanercept significantly blocked thisreduction. These data for the first time links one specific substance,TNF-alpha, to the nucleus pulposus-induced nerve injury.

Example 3

CDP-571 (HUMICADE®)

A 43-year old man with radiating pain corresponding to the left 4thlumbar nerve root is diagnosed as having sciatica with nerve rootdisturbance. He will be treated with 10 mg/kg of CDP-571 (HUMICADE®)intravenously in a single dose.

Example 4

A 38-year old female with radiating pain and slight nerve dysfunctioncorresponding to the 1st sacral nerve on the left side is diagnosed ashaving a disc herniation with sciatica. She will be treated with anintravenous injection of 5 mg/kg of D2E7.

Example 5

CDP-870

A 41-year old female with dermatomal pain corresponding to the firstsacral nerve root on the left side is examined revealing no neurologicaldeficit but a positive straight leg raising test on the left side. Shewill be treated with an intravenous injection of 5 mg/kg of CDP-870.

Example 6

Combination Treatment With a TNF Inhibitor and an IL-1 Inhibitor

Fourteen pigs, (body weight 25-30 kg) received an intramuscularinjection of 20 mg/kg body weight of KETALAR® (ketamine 50 mg/ml,Parke-Davis, Morris Plains, N.J.) and an intravenous injection of 4mg/kg body weight of HYPNODIL® (methomidate chloride 50 mg/ml, AB Leo,Helsingborg, Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon 2mg/ml, Janssen Pharmaceutica, Beerse, Belgium). Anesthesia wasmaintained by additional intravenous injections of 2 mg/kg body weightof HYPNODIL® and 0.05 mg/kg body weight of STRESNIL®. The pigs alsoreceived an intravenous injection of 0.1 mg/kg of STESOLID NOVUM®(Diazepam, Dumex, Helsingborg) after surgery.

Nucleus pulposus was harvested from the 5th lumbar disc through aretroperitoneal approach. Approximately 40 mg of the nucleus pulposuswas applied to the sacrococcygeal cauda equina through a midlineincision and laminectomy of the first coccygeal vertebra. In 5 pigs, thenucleus pulposus was mixed with 100 .mu.g of an anti-TNF-alpha antibody(anti-pig TNF-alpha monoclonal purified antibody, Endogen, Woburn,Mass., USA, Ordering Code MP-390) before application. In five pigs, thenucleus pulposus was mixed with both 100 .mu.g of an anti-TNF-alphaantibody and 100 .mu.g of an anti-IL-1.beta. antibody (anti-pigIL-1.beta. monoclonal purified antibody, Endogen, Woburn, Mass., USA,Catalog No. MP-425). In the remaining 4 pigs, 2 mg of doxycycline(DOXYFERM®, doxycycline 20 mg/ml, Nordic, Malmo, Sweden) was mixed withthe nucleus pulposus before application.

Three days after the application, the pigs were reanaesthetized by anintramuscular injection of 20 mg/kg body weight of KETALAR® and anintravenous injection of 35 mg/kg body weight of PENTOTHAL® (Thiopentalsodium, Abbott lab, Chicago, Ill.). The pigs were ventilated on arespirator. Anesthesia was maintained by an intravenous bolus injectionof 100 mg/kg body weight of Chloralose (alpha)-D(+)-glucochlor-alose,Merck, Darmstadt, Germany) and by a continuous supply of 30 mg/kg/hourof Chloralose. A laminectomy from the 4th sacral to the 3rd coccygealvertebra was performed. The nerve roots were covered with SPONGOSTANE®(Ferrosan, Denmark). Local tissue temperature was continuously monitoredand maintained at 37.538.0. degree. C. by means of a heating lamp.

The cauda equina was stimulated by two E2 subdermal platinum needleelectrodes (Grass Instrument Co., Quincy, Mass.) which were connected toa Grass SD9 stimulator (Grass Instrument Co., Quincy, Mass.) and gentlyplaced intermittently on the cauda equina first 10 mm cranial and then10 mm caudal to the exposed area. To ensure that only impulses fromexposed nerve fibers were registered, the nerve root that exited fromthe spinal canal between the two stimulation sites were cut. An EMG wasregistered by two subdermal platinum needle electrodes, which wereplaced into the paraspinal muscles in the tail approximately 10 mmapart. This procedure is reproducible and represents a functionalmeasurement of the motor nerve fibers of the cauda equina nerve roots.The EMG was visualized using a Macintosh IIci computer provided withSuperscope software and MacAdios II AID converter (GW Instruments,Sommerville, Mass.). The separation distance between the first peaks ofthe EMG from the two recordings was determined and the separationdistance between the two stimulation sites on the cauda equina wasmeasured with calipers. The nerve conduction velocity between the twostimulation sites could thus be calculated from these two measurements.

The person performing the neurophysiologic analyzes was unaware of theexperimental protocol for the individual animal, and after finishing thecomplete study the data were arranged in the three experimental groupsand statistical differences between the groups were assessed byStudent's t-test. For comparison, data from a previous study of theeffects of application of retroperitoneal fat and autologous nucleuspulposus were included (Olmarker K, Rydevik B, Nordborg C, Autologousnucleus pulposus induces neurophysiologic and histologic changes inporcine cauda equina nerve roots, Spine 1993; 18(11): 1425-32).

The average nerve conduction velocity for the five groups is displayedin Table 3 below. According to the previous study, baseline nerveconduction velocity (fat) was 83 m/s and application of autologousnucleus pulposus induced a reduction of the conduction velocity to 45m/s (Olmarker K, Rydevik B, Nordborg C, Autologous nucleus pulposusinduces neurophysiologic and histologic changes in porcine cauda equinanerve roots, Spine 1993; 18(11): 1425-32). Application of doxycycline tothe nucleus pulposus reduced the effects of the nucleus pulposus.Addition of an anti-TNF antibody was more efficient in reducing thenucleus pulposus effects than doxycycline. However, most efficient inblocking the nucleus pulposus induced reduction in nerve conductionvelocity was the combined action of the anti-TNF and the anti-IL-1antibody.

TABLE 3 Nerve conduction velocity (m/s) 3 days after application Fat (n= 5)) 83 +/− 4  NP + doxycycline (n = 4) 53 +/− 22 NP + anti-TNF (n = 5)64 +/− 28 NP + anti-TNF and anti-IL-1-beta (n = 5) 74 +/− 25 NP (n = 5)45 +/− 16

Example 7

Combination Treatment With a TNF Inhibitor and an IL-1 Inhibitor

Seven pigs, (body weight 25-30 kg) received an intramuscular injectionof 20 mg/kg body weight of KETALAR-1® (ketamine 50 mg/ml, Parke-Davis,Morris Plains, N.J.) and an intravenous injection of 4 mg/kg body weightof HYPNODIL® (methomidate chloride 50 mg/ml, AB Leo, Helsingborg,Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon 2 mg/ml,Janssen Pharmaceutica, Beerse, Belgium). Anesthesia was maintained byadditional intravenous injections of2 mg/kg body weight of HYPNODIL® and0.05 mg/kg body weight of STRESNIL®. The pigs also received anintravenous injection of 0.1 mg/kg of STESOLID NOVUM® (Diazepam, Dumex,Helsingborg) after surgery.

Nucleus pulposus was harvested from the 5th lumbar disc through aretroperitoneal approach. Approximately 40 mg of the nucleus pulposuswas applied to the sacrococcygeal cauda equina through a midlineincision and laminectomy of the first coccygeal vertebra. In 2 pigs, thenucleus pulposus was mixed with 100 .mu.g of an antiTNF-alpha antibody(anti-pig TNF-alpha monoclonal purified antibody, Endogen, Woburn,Mass., USA, Ordering Code MP-390) before application. In 2 pigs, thenucleus pulposus was mixed with 100 .mu.g of an anti-IL-1.beta. antibody(anti-pig IL-1.beta. monoclonal purified antibody, Endogen, Woburn,Mass., USA, Catalog No. MP-425). In five pigs, the nucleus pulposus wasmixed with both 100 .mu.g of an anti-TNF-alpha antibody and 100 .mu.g ofan anti-IL-1.beta. antibody.

Seven days after the application, the pigs were reanaesthetized by anintramuscular injection of 20 mg/kg body weight of KETALAR-1® and anintravenous injection of 35 mg/kg body weight of PENTOTHAL® (Thiopentalsodium, Abbott lab, Chicago, Ill.). The pigs were ventilated on arespirator. Anesthesia was maintained by an intravenous bolus injectionof 100 mg/kg body weight of Chloralose (alpha)-D(+)-glucochlor-alose,Merck, Darmstadt, Germany) and by a continuous supply of 30 mg/kg/hourof Chloralose. A laminectomy from the 4th sacral to the 3rd coccygealvertebra was performed. The nerve roots were covered with SPONGOSTANE®(Ferrosan, Denmark). Local tissue temperature was continuously monitoredand maintained at 37.538.0. degree. C. by means of a heating lamp.

The cauda equina was stimulated by two E2 subdermal platinum needleelectrodes (Grass Instrument Co., Quincy, Mass.) which were connected toa Grass SD9 stimulator (Grass Instrument Co., Quincy, Mass.) and gentlyplaced intermittently on the cauda equina first 10 mm cranial and then10 mm caudal to the exposed area. To ensure that only impulses fromexposed nerve fibers were registered, the nerve root that exited fromthe spinal canal between the two stimulation sites were cut. An EMG wasregistered by two subdermal platinum needle electrodes, which wereplaced into the paraspinal muscles in the tail approximately 10 mmapart. This procedure is reproducible and represents a functionalmeasurement of the motor nerve fibers of the cauda equina nerve roots.The EMG was visualized using a Macintosh IIci computer provided withSuperscope software and MacAdios II AID converter (GW Instruments,Sommerville, Mass.). The separation distance between the first peaks ofthe EMG from the two recordings was determined and the separationdistance between the two stimulation sites on the cauda equina wasmeasured with calipers. The nerve conduction velocity between the twostimulation sites could thus be calculated from these two measurements.

The person performing the neurophysiologic analyzes was unaware of theexperimental protocol for the individual animal, and after finishing thecomplete study the data were arranged in the three experimental groupsand statistical differences between the groups were assessed byStudent's t-test. The local animal research ethics committee approvedthe experimental protocol for this experiment. For comparison, data froma previous study of the effects of application of retroperitoneal fatand autologous nucleus pulposus were included (Olmarker K, Rydevik B,Nordborg C, Autologous nucleus pulposus induces neurophysiologic andhistologic changes in porcine cauda equina nerve roots, Spine 1993;18(11): 1425-32). The average nerve conduction velocity for the fivegroups is displayed in the Table 4 below. According to the previousstudy, baseline nerve conduction velocity (fat) was 76 m/s andapplication of autologous nucleus pulposus induced a reduction of theconduction velocity to 45 m/s (Olmarker K, Rydevik B, Nordborg C,Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18(11):1425-32). Application of anti IL-1 showed a similar NCV as NP alone,indicating that the IL-1 had could not reduce the nucleuspulposus-induced reduction in nerve conduction velocity. Application ofanti TNF limited the nucleus pulposus-induced reduction in nerveconduction velocity. Most efficient in reducing the nucleuspulposus-induced reduction in nerve conduction velocity was thecombination of anti IL-1 and anti TNF. Since the effect in limiting thereduction in NCV was more than could be anticipated for addition of theeffects of anti IL-1 and anti TNF, the data indicate a synergisticeffect of the combination of the two cytokine antagonists.

TABLE 4 Nerve conduction velocity (m/s) 7 days after application Fat (n= 5)) 76 +/− 11 NP + anti-IL-1 beta (n = 2) 46 +/− 11 NP + anti-TNF (n =2) 59 +/− 6  NP + anti-TNF and anti-IL-1-beta (n = 3) 78 +/− 2  NP (n =5) 45 +/− 19

The amplitude of the obtained muscle action potentials (MAP's) grosslyreflects the number of conducting axons. The amplitudes afterapplication of the cytokine inhibitors were measured and compared toprevious data of application of annulus fibrosus (NCV 75.+−.11 m/s) andnucleus pulposus at ph 6.0 (NCV 55.+−.22 m/s) from a previous study(Iwabuchi M, Rydevik B, Kikuchi S, Olmarker K, Effects of annulusfibrosus and experimentally degenerated nucleus pulposus on nerve rootconduction velocity, Spine 2001; 26(15): 1651-5) and presented in table5. Amplitudes of MAP seven days after application of cultured nucleuspulposus cells (NCV 52.+−.14 m/s) were also used as reference andexample of the MAP amplitude in NP-exposed nerve roots (Kayama S,Olmarker K, Larsson K, Sjogren-Jansson E, Lindahl A, Cultured,autologous nucleus pulposus cells induce functional changes in spinalnerve roots, Spine 1998; 23(20): 2155-8). NP at pH 6.0 was obtained byslightly lowering the pH to 6.0 by addition of sodium lactate to form a15 mmol/L lactate concentration and to adjusting the pH to 6.0 by addinga 2% HCl solution. The nerve recordings in the NP at pH 6.0 series wereperformed 3 days after application. The difference between applicationof NP at 6.0 and the combination of an anti-TNF and an anti-IL-1inhibitor was statistically significant using Students t-test(p=0.0384), whereas the difference between NP at 6.0 and the twoinhibitors alone were not. Similarly, the difference between applicationof NP cells and the combination of an anti-TNF and an anti-IL-1inhibitor was statistically significant using Students t-test(p=0.0426), whereas the difference between NP cells and the twoinhibitors alone were not. Since the effect in limiting the MAPamplitude was more than could be anticipated for addition of the effectsof anti IL-1 and anti TNF, the data regarding MAP amplitude alsoindicate a synergistic effect of the combination of the two cytokineantagonists.

TABLE 5 Muscle action potential amplitude (m/volts) Annulus fibrosus (n= 5)) 6.5 +/− 2.6 NP + anti-IL-1 beta (n = 2) 2.0 +/− 1.8 NP + anti-TNF(n = 2) 4.1 +/− 0.4 NP + anti-TNF and anti-IL-1-beta (n = 3) 6.1 +/− 2.6NP at pH 6.0 (n = 5) 2.9 +/− 1.0 NP cells (n = 5) 2.8 +/− 1.2

1. A method of treating or alleviating one or more symptoms of a nervedisorder mediated by nucleus pulposus in a mammal comprisingadministering to the mammal a therapeutically effective amount of anantibody that blocks TNF-a activity, wherein the antibody is selectedfrom the group consisting of murine monoclonal antibodies, chimericantibodies, humanized antibodies and human monoclonal antibodies.
 2. Themethod of claim 1, wherein the antibody that blocks TNF-α activity is acomplete or intact antibody.
 3. The method of claim 1, wherein theantibody that blocks TNF-α activity is selected from the groupconsisting of infliximab, CDP-571, D2E7 and CDP-870.
 4. The method ofclaim 1, wherein the antibody that blocks TNF-α activity comprises animmunogenic fragment of an antibody which binds to an epitope of TNF-α.5. The method of claim 4, wherein the immunogenic fragment is selectedfrom the group consisting of Fab fragments, scFv, and F(ab)2 fragments.6. The method of claim 1, wherein the antibody is administeredsystemically.
 7. The method of claim 1, wherein the antibody isadministered orally.
 8. The method of claim 1, wherein the antibody isadministered intramuscularly.
 9. The method of claim 1, wherein theantibody is administered intravenously.
 10. The method of claim 1,wherein the antibody is administered locally.
 11. The method of claim 1,wherein the antibody is administered epidurally.
 12. The method of claim1, wherein the nerve disorder involves pain.
 13. The method of claim 1,wherein the nerve disorder is a nerve root injury.
 14. The method ofclaim 1, wherein the nerve disorder presents as sciatica.
 15. The methodof claim 1, wherein the nerve disorder is caused by a herniated disc.16. A method of treating or alleviating one or more symptoms of aherniated disc in a mammal comprising administering to the mammal atherapeutically effective amount of an antibody that blocks TNF-αactivity, wherein the antibody is selected from the group consisting ofmurine monoclonal antibodies, chimeric antibodies, humanized antibodiesand human monoclonal antibodies.
 17. The method of claim 16, wherein theantibody that blocks TNF-a activity is a complete or intact antibody.18. A method of treating or alleviating one or more symptoms of sciaticain a mammal comprising administering to the mammal a therapeuticallyeffective amount of an antibody that blocks TNF-α activity, wherein theantibody is selected from the group consisting of murine monoclonalantibodies, chimeric antibodies, humanized antibodies and humanmonoclonal antibodies.
 19. The method of claim 18, wherein the antibodythat blocks TNF-α activity is a complete or intact antibody.
 20. Themethod of claim 16 or 18, wherein the antibody that blocks TNF-αactivity is selected from the group consisting of infliximab, CDP-571,D2E7 and CDP-870.
 21. The method of claim 16 or 18, wherein the antibodythat blocks TNF-α activity comprises an immunogenic fragment of anantibody which binds to an epitope of TNF-α.
 22. The method of claim 18,wherein the immunogenic fragment is selected from the group consistingof Fab fragments, scFv, and F(ab)2 fragments.
 23. The method of claim 16or 18, wherein the antibody is administered locally.
 24. The method ofclaim 16 or 18, wherein the antibody is administered epidurally.